Abstract
The successful medical application of single domain antibodies largely depends on their functionality. This feature is partly determined by the intrinsic stability of the single domain. Therefore a lot of research has gone into the elucidation of rules to uniformly increase stability of antibodies. Recently, a novel intra-domain disulfide bond was independently discovered by two research groups, after either rational design or careful investigation of the naturally occurring camelid antibody repertoire. By introducing this particular disulfide bond within a single domain antibody, the conformational stability can be increased in general. In this chapter it is described how to introduce this extra intra-domain disulfide bond and how to estimate the biophysical and biochemical impact of this cystine on the domain.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K (2009) General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem 284:3273–3284
Jespers L, Schon O, Famm K, Winter G (2004) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165
Famm K, Hansen L, Christ D, Winter G (2008) Thermodynamically stable aggregation-resistant antibody domains through directed evolution. J Mol Biol 376:926–931
Saerens D, Pellis M, Loris R, Pardon E, Dumoulin M, Matagne A, Wyns L, Muyldermans S, Conrath K (2005) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 352:597–607
Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, Muyldermans S, Wyns L, Matagne A (2002) Single-domain antibody fragments with high conformational stability. Protein Sci 11:500–515
van der Linden RH, Frenken LG, de Geus B, Harmsen MM, Ruuls RC, Stok W, de Ron L, Wilson S, Davis P, Verrips CT (1999) Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta 1431:37–46
Ladenson RC, Crimmins DL, Landt Y, Ladenson JH (2006) Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem 78:4501–4508
Ewert S, Cambillau C, Conrath K, Pluckthun A (2002) Biophysical properties of camelid V(HH) domains compared to those of human V(H)3 domains. Biochemistry 41:3628–3636
Perez JM, Renisio JG, Prompers JJ, van Platerink CJ, Cambillau C, Darbon H, Frenken LG (2001) Thermal unfolding of a llama antibody fragment: a two-state reversible process. Biochemistry 40:74–83
Hagihara Y, Matsuda T, Yumoto N (2005) Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains. J Biol Chem 280:24752–24758
van der Vaart JM, Pant N, Wolvers D, Bezemer S, Hermans PW, Bellamy K, Sarker SA, van der Logt CP, Svensson L, Verrips CT, Hammarstrom L, van Klinken BJ (2006) Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments. Vaccine 24:4130–4137
Hagihara Y, Mine S, Uegaki K (2007) Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region. J Biol Chem 282:36489–36495
Saerens D, Conrath K, Govaert J, Muyldermans S (2008) Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains. J Mol Biol 377:478–488
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Lab Press, Plainview
Weiner MP, Costa GL, Schoettlin W, Cline J, Mathur E, Bauer JC (1994) Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151:119–123
Barik S (1997) Mutagenesis and gene fusion by megaprimer PCR. Methods Mol Biol 67:173–182
Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 33:1033–1038
Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954
Ellman GL (1958) A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys 74:443–450
Bates RG, Robinson RA (1973) Tris (hydroxymethyl) aminomethane. Useful secondary pH standard. Anal Chem 45:420
Fukada H, Takahashi K (1998) Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride. Proteins 33:159–166
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
Eyer P, Worek F, Kiderlen D, Sinko G, Stuglin A, Simeon-Rudolf V, Reiner E (2003) Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal Biochem 312:224–227
Riddles PW, Blakeley RL, Zerner B (1983) Reassessment of Ellman’s reagent. Methods Enzymol 91:49–60
Riddles PW, Blakeley RL, Zerner B (1979) Ellman’s reagent: 5,5′-dithiobis(2-nitrobenzoic acid)–a reexamination. Anal Biochem 94:75–81
Kabat EA, Wu TT, Perry HM, Gottsman KS, Foeller C (1991) Sequences of proteins of immunologic interest, 5th edn. Public Health Service, National Institutes of Health, US Department of Health and Human Services, Bethesda
Acknowledgements
No financial interest is declared by the authors. We would like to thank Ms. Kaede Lilian-Komaba, Ir. Jochen Govaert, and Dr. Ir. Cécile Vincke for critically reading the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hagihara, Y., Saerens, D. (2012). Improvement of Single Domain Antibody Stability by Disulfide Bond Introduction. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_24
Download citation
DOI: https://doi.org/10.1007/978-1-61779-968-6_24
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-967-9
Online ISBN: 978-1-61779-968-6
eBook Packages: Springer Protocols